Figure 3: Dynasore-mediated inhibition of VEGF-to-ERK1/2 signalling is independent of dynamin.

(a) HUVECs transduced with lentiviral vectors encoding dynamin (1 and 2) or dynamin K44A (1 and 2) were stimulated with VEGF for various time points, lysed and subjected to immunoblotting analysis using antibodies against the phosphorylated or total ERK1/2. Quantification of the effect of dynamin inhibition on the VEGF-induced phosphorylation of ERK1/2 is shown on the right of the immunoblots. The effect is considered non-significant (n = 3, mean ± S.D., ANOVA followed by Dunnett’s analysis). (b) Dynamin2 siRNAs treated HUVECs were serum starved for 2 h, incubated with 100 μM dynasore for 30 min and stimulated with VEGF for 10 min, lysed and processed for immunoblotting analysis using antibodies against the phosphorylated and total forms of ERK1/2. Immunoblots are representative of 3 independent experiments. Quantification is shown on the right of the immunoblots (n = 3, mean ± S.D., ***P < 0.001, ANOVA followed by Dunnett’s analysis). The efficiency of dynamin2 knockdown was assessed by semi-quantitative RT-PCR (see agarose gel at the bottom of b), as well as by immunofluorescence microscopy analysis of transferrin uptake (see S1b).